Amebiasis and related vision-threatening infections caused by amoebae is a major contributor to diarrheal diseases, primary amoebic meningoencephalitis and cornea problems. Sequencing of amoeba genomes has evoked a broad search for new, pathogen-specific drug targets, of which the sterol C24-methyltransferase (24-SMT) is a clear candidate since it is synthesized in protozoa but not in animals. Notably, 24-SMT is responsible for the introduction of the methyl group at C24 into the ergosterol side chain. The cholesterol side chain is missing this structural feature which is crucial to ergosterol function. In the R21 phase, we will incubate a series of mechanism-based inhibitors of 24-SMT that differ in the sterol frame and electronics of the side chain with cultured cells of Acanthamoeba and Naegleria and determine which compounds are the most potent inhibitors of ergosterol biosynthesis, trophozoites growth and cyst formation. Additionally, to make improvements to existing drug therapy for treating amoeba infections we will further evaluate in vitro representative anti-fungal azoles that target sterol 14-demethylase (14-SDM; CYP51). Therefore, we will characterize the substrate preference and product outcome of cloned enzymes and use these enzymes to determine inhibitor specificity, binding and covalent inactivation properties. The R33 phase will be undertaken with the proof-of-concept demonstrated. We will test in vitro whether our lead molecules that inhibit 24-SMT in combination with traditional chemotherapeutics have synergistic activities. Additionally, in collaboration with investigators at Meharry Medical College and UTSouthwestern Medical Center, we will evaluate our lead candidate drugs in a mouse model of Acanthamoeba keratitis or primary amebic meningoencephalitis due to Naegleri fowleri. With the aid of a collaborator at Texas Tech University Health Sciences Center, crystal structures of 24-SMT and 14- SDM complexed with relevant inhibitors will be generated to identify binding sites with certainty and should reveal interactions involved with catalysis. ADME/toxicity properties will be evaluated by the bioavailability and metabolism of 3H-inhibitor fed to healthy mice. The overall goal of these studies is to establish mechanism-based inhibitors as a novel class of anti- amoeba agents and to develop synergistic partners of steroidal inhibitors and antifungal agents (medical azole or amphotericin B) that target ergosterol biosynthesis and processing. Specific therapeutic combinations of these compounds could achieve optimal amebacidal effectiveness that thereby, provide for better healthcare.